Methods for DNA amplificating and sequencing

ABSTRACT

The osmoprotectants proline, 2-methyl-4-carboxy-3,4,5,6-tetrahydropyrimidine (“THP(B)”, and 2-methyl-4-carboxy-5-hydroxy-3,4,5,6,-tetrahydropyrimidine (“THP(A)”) are capable of increasing the thermal stability of DNA polymerases at elevated temperatures. THP(B) is further effective in lowering the melting temperature of double-stranded DNA. Proline, THP(A) and THP(B) are thus useful in procedures involving melting of double-stranded DNA and/or polymerase-mediated DNA synthesis, such as in primer extension, in PCR (polymerase chain reaction) amplification and in DNA sequencing.

CROSS REFERENCE TO RELATED APPLICATION

The present application is the national stage under 35 U.S.C. 371 ofPCT/IL99/00080, filed Feb. 8, 1999.

FIELD OF THE INVENTION

The present invention relates to a method for performing cycled primerextension on a DNA template, and more particularly to methods includinga primer extension step such as polymerase chain reaction (PCR)amplification and nucleotide sequencing comprising performing said PCRand sequencing reactions in the presence of an osmoprotectant selectedfrom proline 2-methyl-4-carboxy-3,4,5,6-tetrahydropyrimidine(hereinafter “THP(B)”),2-methyl-4-carboxy-5-hydroxy-3,4,5,6-tetrahydropyrimidine (hereinafter“THP(A)”) and mixtures thereof, to improve yield and specificity of saidreactions. The invention further relates to kits comprising proline,THP(B), THP(A), or mixtures thereof for use in PCR amplification and incycle nucleotide sequencing.

BACKGROUND OF THE INVENTION

Primer extension on a DNA template is a step common to some of the mostuseful and powerful techniques in molecular biology. Polymerase chainreaction (PCR), one of these techniques, is a rapid, inexpensive andsimple means of producing microgram amounts of DNA from minutequantities of source materials. Many variations on the basic procedurehave now been described and applied to a range of disciplines.

In medicine, PCR's major impact is on the diagnosis and screening ofgenetic diseases and cancer, the rapid detection of mycobacteria andHIV, the detection of minimal residual disease in leukemia, and HLAtyping. The PCR technique is also useful in forensic pathology andevolutionary biology, plays a central role in the human genome projectand is routinely used in molecular biology processes (McPherson et al.,1992).

However, the practical use of PCR technology frequently facesdifficulties and limitations. The necessity to convert originally duplexsource DNA and then double-stranded DNA products into single strandedtemplates in every cycle of amplification is normally accomplished bythermal denaturation of DNA at 93-95° C. The DNA denaturation greatlydepends on its nucleic base composition. A high GC content renders DNAamplification and sequencing very difficult, due to increased meltingtemperature and the stable secondary structure of the expanded motif. Acommon result of amplifying a region containing a repeat motif with ahigh GC content is the presence of additional amplification products,which do not correspond to the desired product (Varadaraj and Skinner,1994). In addition, incomplete denaturation allows DNA strands to “snapback”, leading to a decrease in product yield. Denaturation steps thatare conducted for long periods of time and/or at a high temperature leadto unnecessary loss of enzyme activity and dNTP decomposition.

Taq DNA polymerase, ordinarily used in PCR protocols, can withstandrepeated exposure to the high temperature (94-95° C.) required fortypical DNA strand separation, and thus simplifies the PCR procedure byeliminating the need to add an enzyme in each cycle. However, Taqpolymerase appears to extend a mismatched primer/template in comparisonto other polymerases with proofreading exonuclease activities, e.g.Klenow and T7 DNA polymerases, which are non-thermostable.

Another very effective technique employing primer extension is the cyclesequencing technique used for determining the order of nucleic acids ina target nucleotide sequence. This procedure involves repeated cycles ofprimer extension while the target nucleotide sequence is sequenced.

Similar considerations, as mentioned above for the PCR method, apply forthe cycle sequencing procedure. In sequencing reactions as well, thecomplete denaturation of the template DNA is of crucial importance for asuccessful reaction. Thus, regions of DNA with repeat motifs, high GCcontent and rigid secondary structures are difficult to sequence. Inaddition, sequencing of a very long stretch of nucleotides, or of atarget nucleotide sequence present in a minute amount is problematic.The ability to accomplish a complete denaturation of double stranded DNAand to perform sequencing reactions at reduced temperatures, either withTaq polymerase or with non-thermostable polymerase, is advantageous interms of both yield and accuracy.

In an attempt to improve the yield and specificity of PCR and sequencingreactions, a number of buffer additives were employed. It was shown thatcertain cosolvents, such as DMSO (Pomp and Medrano, 1991; Filichkin andGelvin, 1992), glycerol (Cheng et al., 1994; U.S. Pat. Nos. 5,432,065and 5,545,539), formamide (Comey et al., 1991) and betaine (GermanPatents DE 4411594 C1 and DE 4411588 C1; Mytelka et al., 1996),facilitate standard PCR and/or cycle sequencing. It has been suggestedthat DMSO may affect the melting temperatures (Tm) of the template DNAand of the oligonucleotide primers and/or the degree of product strandseparation at a particular “denaturation” as well as improving thethermal activity of Taq DNA polymerase (Gelfand and White, 1989).Glycerol may influence long amplifications by (i) doubling the thermalstability of Taq polymerase at 95-97° C., and (ii) effectively loweringDNA melting temperatures (by 2.5-3° C. for each 10% increase in glycerolconcentration) (Cheng et al., 1994). Yet, the use of these bufferadditives is limited, e.g. solutions containing glycerol in effectiveconcentrations of 20-40% are viscous and difficult to handle (U.S. Pat.No. 5,432,065), DMSO in 10% concentration inhibits Taq DNA polymeraseactivity by 53% and T7 DNA polymerase is completely inactive in 40%formamide.

The compounds 2-methyl-4-carboxy-3,4,5,6-tetrahydropyrimidine [THP(B)],also known as ectoine, and its hydroxy derivative,2-methyl-4-carboxy-5-hydroxy-3,4,5,6-tetrahydropyrimidine [THP(A)] werepreviously identified in the laboratory of the inventors of the presentinvention as metabolites in several Streptomyces microorganisms (Inbarand Lapidot, 1988a; 1988b and 1991; Malin and Lapidot, 1996). Ectoinewas also found in a variety of halophilic and halotolerant bacteria(Galinski et al., 1985). THP(B) and THP(A) are zwitterionic compounds(Inbar et al. 1993; FIG. 1) with many useful properties such asosmoprotection and thermoprotection of several organisms of theStreptomyces species and E. coli cells (Malin and Lapidot, 1996). THP(B)and THP(A) are not toxic neither to mammalian cells nor to animals(Lapidot et al., 1995). Israel patent No. 100810 and corresponding U.S.Pat. No. 5,789,414 and European Patent No. EP 0553884 of the presentapplicants disclose that THP(A) and THP(B) interact with and protect DNAin non-tumor tissues from damage by DNA-binding drugs and thus can beused for decreasing the toxic effects of DNA-binding drugs such asadriamycin and actinomycin D.

Proline is another osmoprotectant that accumulates in plants, bacteria,algae and marine invertebrates as a response to salinity stress. Prolinewas shown to destabilize DNA and to partially counteract the effect ofsodium chloride and spermidine on the stability of the double helix, andto lower the melting temperature of DNA in a concentration-dependentmanner (Rajendrakumar et al., 1997).

None of the above references describes or suggests the use of proline,THP(A) or THP(B) or mixtures thereof as additives to PCR reactionmixtures and in reactions for nucleotide sequencing.

SUMMARY OF THE INVENTION

It has now been found, according to the present invention, that THP(B)is effective in lowering the melting temperature of double-stranded DNA,and that proline, THP(B) and THP(A) are capable of increasing thethermal stability of DNA polymerases at elevated temperatures,indicating that they can be useful in procedures involving melting ofdouble-stranded DNA and/or polymerase-mediated DNA synthesis, such as inprimer extension, in PCR (polymerase chain reaction) amplification andin DNA sequencing.

Thus, in one aspect, the present invention provides a method forperforming a cycled primer extension reaction comprising the steps of:

(i) contacting a template DNA comprising a target sequence ofnucleotides with at least one primer oligonucleotide complementary to anucleotide sequence at the 3′-end of said target sequence, underconditions allowing annealing of said primer to its complementarynucleotide sequence on said target sequence, in the presence of anosmoprotectant selected from proline,2-methyl-4-carboxy-3,4,5,6-tetrahydropyrimidine [THP(B)] and mixturesthereof, to lower the melting temperature of said template DNA and/or ofsaid primer; and

(ii) carrying out a polymerase-mediated extension of said primer on saidtarget sequence of nucleotides in the presence of an osmoprotectantselected from proline, THP(B),2-methyl-4-carboxy-5-hydroxy-3,4,5,6-tetrahydropyrimidine [THP(A)] andmixtures thereof, to stabilize said polymerase,

thus obtaining a high yield specific extension of the primer on saidtarget sequence of nucleotides of the template DNA.

Steps (i) and (ii) may be repeated a plurality of times, for example10-90 times, preferably 15-35 times, and each step (i) is preceded byDNA thermal denaturation at a temperature suitable for separating bothsaid template DNA into its strands and the polymerase-extended primer ofstep (ii) from its complementary target sequence of nucleotides, saidtemperature being a temperature in which the polymerase used in step(ii) is stable.

In one embodiment, the invention relates to a method for determining anucleotide sequence of a target DNA, wherein in step (i) the targetsequence of the template DNA is a sequence of nucleotides to besequenced, and the polymerase-mediated extension of the primer in step(ii) is carried out in the presence of all four dNTPs: dATP, dCTP, dGTPand dTTP, and in the presence of a minute amount of either ddATP, ddCTP,ddGTP or ddTTP, prior to the determination of the nucleotide sequence ofthe target DNA. The dGTP can be substituted by 7-deaza-dGTP described inEP 0212536.

According to this embodiment, the method for determining a nucleotidesequence of a target DNA comprises the steps of:

(i) heating a template DNA comprising a target sequence of nucleotidesto be sequenced at a temperature suitable for separating said templateDNA into its strands in the presence of an osmoprotectant selected fromproline, 2-methyl-4-carboxy-3,4,5,6-tetrahydropyrimidine [THP(B)] andmixtures thereof;

(ii) contacting said denatured template DNA of step (i) with a primeroligonucleotide complementary to a nucleotide sequence at the 3′-end ofsaid target sequence of nucleotides under conditions allowing annealingof said primer to its complementary nucleotide sequence on the targetsequence, in the presence of an osmoprotectant selected from proline,2-methyl-4-carboxy-3,4,5,6-tetrahydropyrimidine [THP(B)] and mixturesthereof;

(iii) carrying out a polymerase-mediated extension of said primer ofstep (ii) in the presence of all four natural dNTPs: dATP, dCTP, dGTP(or 7-deaza-dGTP) and dTTP, of a minute amount of either ddATP, ddCTP,ddGTP or ddTTP and of an osmoprotectant selected from proline, THP(B),2-methyl-4-carboxy-5-hydroxy-3,4,5,6-tetrahydropyrimidine [THP(A)] andmixtures thereof;

(iv) repeating steps (i)-(iii) a plurality of times; and

(v) determining the nucleotide sequence of the target DNA.

In another embodiment, the invention provides a method for amplifying atarget sequence of nucleotides by polymerase chain reaction (PCR),wherein in step (i) the target sequence of the template DNA is asequence of nucleotides to be amplified and the template DNA iscontacted with two oligonucleotide primers complementary to thenucleotide sequences at the 3′-ends of said target sequence ofnucleotides and its opposite strand; in step (ii) a polymerase-mediatedextension of the annealed primers of step (i) is carried out; steps(i)-(ii) are repeated a plurality of times, the last step being step(ii), thus generating multiple copies of the target sequence ofnucleotides.

According to this embodiment, the invention relates to a method foramplifying a target sequence of nucleotides by polymerase chain reaction(PCR) comprising the steps of:

(i) heating a template DNA comprising a target sequence of nucleotidesto be amplified at a temperature suitable for separating said templateDNA into its strands in the presence of an osmoprotectant selected fromproline, 2-methyl-4-carboxy-3,4,5,6-tetrahydropyrimidine [THP(B)] andmixtures thereof;

(ii) contacting the template DNA of step (i) with two oligonucleotideprimers complementary to nucleotide sequences at the 3′-ends of saidtarget sequence of nucleotides and its opposite strand; in the presenceof an osmoprotectant selected from proline,2-methyl-4-carboxy-3,4,5,6-tetrahydropyrimidine [THP(B)] and mixturesthereof, under conditions allowing annealing of said oligonucleotideprimers to their complementary sequences on said target sequence ofnucleotides and its opposite strand;

(iii) carrying out a polymerase-mediated extension of the annealedprimers of step (ii) in the presence of an osmoprotectant selected fromproline, THP(B),2-methyl-4-carboxy-5-hydroxy-3,4,5,6-tetrahydropyrimidine [THP(A)] andmixtures thereof; and

(iv) repeating steps (i)-(iii) a plurality of times, the last step beingstep (iii), thus generating multiple copies of the target sequence ofnucleotides.

The methods of the invention are particularly useful for reactionsinvolving GC-rich DNAs, thus diminishing or eliminating the difficultiesfound in amplification and sequencing of GC-rich DNA molecules. Themethods are further useful for reactions involving in step (ii) or (iii)a thermostable DNA polymerase, such as Taq polymerase Klentaqlpolymerase and Pfu polymerase, or a non-thermostable DNA polymerase suchas T7 DNA polymerase, T4 DNA polymerase, Klenow fragment of DNApolymerase I, reverse transcriptases, Bca polymerase, Bst polymerase andmutants of these polymerases.

In another aspect, the invention relates to the use of an osmoprotectantselected from proline, THP(B), THP(A) and mixture thereof as an additivein a reaction for determining a nucleotide sequence or as an additive toa PCR reaction mixture, and to kits comprising in separate containers:(a) the reagents necessary for DNA sequencing or the reagents necessaryfor a polymerase chain reaction, and (b) proline, THP(A) or THP(B).

In a further aspect, the invention relates to a method for lowering themelting temperature of double-stranded DNA (dsDNA) comprising adding tothe incubation mixture of said dsDNA an effective amount of THP(B).

In a further aspect, the invention relates to a method for increasingstability of a DNA polymerase at elevated temperatures comprising addingto the incubation mixture of said polymerase an effective amount of anosmoprotectant selected from proline, THP(B), THP(A) and mixturesthereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the structural formulas of THP(A) (left) and THP(B)(right).

FIG. 2 depicts thermal transition of calf thymus DNA in the absence andpresence of the indicated amounts of THP(B): 0.8M, 2M, 3M and 4M. DNAmelting was performed as described in Materials and Methods, section(ii).

FIG. 3 depicts the variation of melting temperature (Tm) with THP(B)concentration for DNAs of varying base compositions. DNA melting wasperformed and Tms determined as described in Materials and Methods,section (ii). (filled triangles)—Calf thymus DNA; (filledcircles)—Micrococcus lysodeikticus DNA; (filled squares)—Clostridiumperfringens DNA; (open triangles)—poly(dA-dT).

FIGS. 4A-B depict thermal transitions of the oligonucleotides[d(ATGCAT)]₂ (SEQ ID NO: 1) and [d(GCTTAAGC)]₂ (SEQ ID NO: 2),respectively. The chemical shifts of the C4H5 proton of [d(ATGCAT)]₂(SEQ ID NO: 1) and of the G1H8 proton of [d(GCTTAAGC)]₂ (SEQ ID NO: 2)were measured as described in Materials and Methods section (iii), as afunction of increasing temperatures in the absence (open squares) orpresence of 0.5M (open circles) and 1M (filled squares) THP(B).

FIG. 5 depicts the time course of thermal inactivation of Taq DNApolymerase at 97° C. in the absence (filled circles) and presence ofeither 1M THP(B) (filled squares), 1M THP(A) (open squares) or 1Mglycerol (open diamonds). The thermal inactivation was determined atdifferent periods of time from Taq polymerase remaining activitymeasured as described in Materials and Methods, section (vi).

FIG. 6 depicts ethidium bromide staining of PCR-amplified DNA productsrun on 1.7% agarose gel. PCR was performed according to the proceduredescribed in Materials and Methods, section (viii), for amplification ofa 349 b.p. fragment (66.5% GC content) from Halobacterium marismortuigenomic DNA template at two different denaturation temperatures (Td),95° C. (left) and 92° C. (right), in the absence and in the presence of0.5M THP(B), as indicated. Two or three repetitions of each experimentare shown.

FIGS. 7A-7C depict ethidium bromide staining of PCR-amplified DNAproducts run on 1.7% agarose gel. PCR was performed according to theprocedures described in Materials and Methods, section (viii), foramplification of a 349 bp. fragment (66.5% GC content) fromHalobacterium marismortui genomic DNA template at three differentdenaturation temperatures (Td): 95° C. (FIG. 7A), 90° C. (FIG. 7B) and89° C. (FIG. 7C), in the absence and presence of 1.0M THP(B). Two orthree repetitions of each experiment are shown.

FIGS. 8A-8B depict thermal transition of calf thymus DNA (FIG. 8A) andother DNAs (FIG. 8B) in the presence and absence of proline. DNA meltingwas performed as described in Materials and Methods, section (ii). FIG.8A: —(filled diamonds)—no proline added; (open squares)—2.0M; (filledtriangles)—3.5M; (filled squares)—5.0M; (open triangles)—5.5 M; (filledcircles)—6.2M proline. FIG. 8B, in the presence of 6.2 M proline: (opentriangles)—Micrococcus lysodeikticus DNA; (filled triangles)—Clostridiumperfringens DNA; (filled squares)—calf thymus DNA; (filledcircles)—poly(dA-dT).

FIG. 9A depicts a variation of Tm with proline concentration for DNAs ofvarying base compositions. DNA melting was performed as described inMaterials and Methods, section (ii). (filled squares)—calf thymus DNA;(open triangles)—Micrococcus lysodeikticus DNA; (filledtriangles)—Clostridium perfringens DNA; (filled circles)—poly(dA-dT).FIG. 9B depicts changes in dTm/dGC as a function of prolineconcentration.

FIG. 10 depicts Klenow DNA polymerase activity at 37° C. in the absence(dark bars) and in the presence of 5.0M proline (hatched bars). Theactivity of Klenow DNA polymerase was measured at 6.7, 10 and 15 mMMgCl₂ as described in Materials and Methods, section (iv).

FIG. 11 depicts the time course of thermal inactivation of Klenow DNApolymerase at 65° C. in the absence (filled circles) and presence ofeither 5M proline (open diamonds) or 5M glycerol (filled triangles). Theremaining activity of Klenow DNA polymerase was measured at differentperiods of time as described in Materials and Methods, section (vii).

FIG. 12 depicts ethidium bromide staining of PCR-amplified DNA productsrun on 2.0% agarose gel. PCR was performed according to the proceduredescribed in Materials and Methods, section (ix), for amplification of a349 b.p, fragment (66.5% GC) from Halobacterium marismortui genomic DNA,using 10 and 15 units of Klenow fragment of DNA polymerase I.

FIG. 13 depicts ethidium bromide staining of PCR-amplified DNA productsrun on 1.7% agarose gel. PCR was performed according to the proceduredescribed in Materials and Methods, section (x), for amplification of a349 b.p. fragment (66.5% GC) from Halobacterium marismortui genomic DNAtemplate catalyzed by Taq DNA polymerase at two different denaturationtemperatures (Td), at 95° C. and 91° C., in the absence and in thepresence of 1.0M proline. Three repetitions of each experiment areshown.

FIG. 14 depicts ethidium bromide staining of PCR-amplified DNA productsrun on 1.7% agarose gel. PCR was performed according to the proceduredescribed in Materials and Methods, section (xi), for amplification of a349 b.p. fragment (66.5% GC) from Halobacterium marismortui genomic DNAtemplate catalyzed by KlenTaql DNA polymerase at two differentdenaturation temperatures (Td), at 77° C. and 75° C., in the presence of4.0 M proline. Two repetitions of each experiment are shown.

DETAILED DESCRIPTION OF THE INVENTION

The term “primer extension” as used herein in the specification refersto a process of increasing the length of an oligonucleotidecomplementary to a nucleotide sequence comprised within a template DNA.The process consists of repeatedly adding to the oligonucleotide's3′-end a single nucleotide which is dictated by the nucleotide presentat the corresponding position in the complementary template DNA strand.The term “cycled primer extension” refers to a procedure which involvesrepeated cycles in which primer extension is alternated with periodicheating whereby separation of the extended primer from the template DNAstrand occurs.

The term “melting temperature (Tm)” of double-stranded DNA (dsDNA)refers to a temperature at which 50% of a dsDNA sample is separated intoits two complementary DNA strands.

The term “amplifying” refers to repeated copying of a specified sequenceof nucleotides resulting in an increase in the amount of said specifiedsequence of nucleotides.

The term “sequencing” refers to a procedure for determining the order inwhich nucleotides occur in a target nucleotide sequence.

The term “target nucleotide sequence” refers to a nucleotide sequencewhich is intended to be duplicated, amplified or sequenced.

The term “template DNA” refers to DNA molecules or fragments thereof ofany source or nucleotide composition, that comprise a target nucleotidesequence as defined above.

According to the present invention, THP(B) or proline or mixturesthereof can significantly lower the melting temperature of dsDNA, andproline, THP(B) or THP(A), alone or in combination, increase thestability of DNA polymerases incubated at elevated temperatures.

THP(B) and THP(A) for use in the invention can be isolated from naturalsources such as, for example, from actinomycin D-producingmicroorganisms of the Streptomyces species, e.g., S. parvulus, S.chrismomalus, or S. antibioticus, and separated in purified form asdescribed in IL Patent No. 100810 and corresponding U.S. Pat. No.5,789,414 and EP 0553884. THP(B) alone can be isolated and purified fromhalophilic and halotolerant bacteria such as bacteria of the genusEctothiorhodospira, e.g. E. halochloris, E. halophila and mutantsthereof or from heterotrophic halophilic eubacteria of the familyHalomonadaceae grown in high salinity conditions. THP(A) alone can beisolated and purified from soil microorganisms of the Streptomycesspecies, e.g. S. clavuligerus, S. griseus and mutants thereof, under lowsalt stress such as 0.25-0.5M NaCl. THP(B) can also be syntheticallyproduced as described in Japanese Patent Application No. 63-259827.

L-Proline is a common amino acid that is commercially available or canbe synthetically produced and obtained in highly purified form.

According to the invention, THP(B) was found to decrease the Tm ofoligonucleotides as short as 6-8 mers and of dsDNAs being either genomicDNAs, cDNAs or recombinant DNA molecules, in a concentration dependentmanner in concentrations ranging from 0.5 to 4M. The melting temperatureof short oligonucleotides (6 or 8 mers) were reduced by 3 to 6° C. inthe presence of 0.5M and 1.0 M THP(B).

The magnitude of the Tm decrease depends on the GC content of theparticular oligonucleotide or dsDNA, being more pronounced with high GCcontent DNAs. For example, the Tm decrease of calf thymus DNA (42% GC)and of Micrococcus lysodeikticus DNA (72% GC content) in the presence ofTHP(B) was significantly higher than that of Clostridium perfringens DNA(26% GC content), while no change in the Tm of the syntheticoligonucleotide poly(dA-dT) could be observed in the presence of THP(B)at concentrations as high as 4M. At 4.0 M concentration of THP(B), DNAswith different GC content melt in a very narrow temperature range(40-43° C.), while in the absence of THP(B) the melting temperatureranges from 39 to 75° C. Isostabilization of the DNA molecule by THP(B)may be explained as a result of greater destabilization of GC-rich thanAT-rich DNAs. THP(B) eliminates the DNAs base paircomposition-dependence on DNA melting.

Proline, known to decrease DNA melting temperature (Rajendrakumar etal., 1997), was found according to the invention to only slightlydecrease Klenow polymerase enzymatic activity and to be a betterstabilizer of Klenow polymerase than glycerol, with a half-life of theenzyme of 21 min at 65° C. in 5 M L-proline. These findings have enableda successful design of a PCR protocol for a rather GC-rich genomic DNAtemplate. The amount of Klenow polymerase in the herein presentedprotocol (10-15 units) can be further reduced when 7-deaza-dGTP is usedinstead of dGTP, due to the expected decrease of denaturationtemperature. The results herein reveal that proline concentration in therange of 3-5.5 M is sufficient to confer stability to Klenow polymerase.

Proline can be used as a sole additive in the protocol or in combinationwith glycerol or any other DNA-destabilizing agent which the polymerasetolerates, such as THP(B) or THP(A). Proline (up to 5.0 M) decreases themelting temperature (Tm) of various DNAs and leads to DNA partial“isostabilization” (a decrease of Tm difference between GC and AT pairs,manifested by an apparent linear decrease of dTm/GC factor (Melchior etal., 1973; Rees et al., 1993), while at higher concentrations, prolinedestabilizes GC and AT pairs evenly. A complete “isostabilation” of DNA,as in the case of betane (Rees et al., 1993) THP(B) (equal stability ofAT and GC pairs, dTm/dGC=0), was not reached for proline. The Tm valuesof the tested natural DNAs (57-78° C.) decreased to a narrow range of28-32° C. in the presence of 6.2 M prolione. The partial“isostabilization” of DNA by proline at high concentration may cause lowspecificity of PCR, when 20-25 b.p. primers are used.

Primers of 30-35 b.p. length, used in the herein presented PCR protocol,were found to be effective to remedy the decreased priming specificityat high concentrations of proline, and to achieve a good selectivity ofamplification.

Besides standard PCR and DNA sequencing, the protocol with proline canbe interesting for the following methods: a) use of Klenow polymerase incombination with contiguous hexamer primers and single-stranded DNAbinding protein for a specific primer formation (Kieleczawa et al.,1992) utilizing a rather low amount of a source DNA; b) low denaturationtemperature cycling might enable usage of less thermostable labels forDNA sequencing or PCR. This approach might be useful for otherthermolabile polymerases in PCR and other DNA amplification methods. Forexample, T7 DNA polymerase and its modifications, able to amplifyGC-rich DNA and regions with stable secondary structures, could providesolutions to the cases still remaining beyond today's practical PCR andDNA sequencing capabilities, such as amplification of long CGG tripletrepeat sequences. Introduction of T4 polymerase to cycled PCR might beof interest for the cases requiring high fidelity, e.g. foramplification of sequences present at a very low frequency requiringmany cycles of amplification to be detected.

According to the invention it was further found that proline, THP(B) andTHP(A), alone or in combination, can stabilize both thermostable andnon-thermostable DNA polymerases incubated at elevated temperatures, thestabilizing effect being more pronounced when the enzyme is incubatedfor prolonged periods of time and at a higher temperature than thetemperature of their optimal activities. The thermostable Taqpolymerase, after 30-35 min incubation under typical DNA denaturationtemperature at 95° C., is only 50% active, and after 30 min incubationat 97° C., only 10% active in comparison to 40% in the presence of 1MTHP(B) and even higher, 55%, in the presence of THP(A). A much moredramatic effect is obtained at longer incubation time (60 min), wherethe remaining activity is less than 5% without additives and is 55% inthe presence of THP(A) (by 10-fold higher). The non-thermostablepolymerases are much more sensitive to thermal inactivation, forexample, the half life of Klenow DNA polymerase is around 30-50 secondsat 65° C., whereas in the presence of 5M proline it is 25 min, about30-50 fold longer.

In preferred embodiments, cycled primer extension of any template DNA isconducted with the thermostable Taq polymerase at 60-80° C. in thepresence of 0.5-3.5M THP(B), optionally with 0.5-3.0M THP(A), or 1-5Mproline, or with a non-thermostable polymerase at 30-65° C. in thepresence of 1-3 M THP(B), optionally with 0.5-3.0M THP(A), or 1-5Mproline.

Lowering the Tm of dsDNA by proline and/or THP(B) and stabilization ofDNA polymerases by proline, THP(B) and/or THP(A) are beneficial forcycled primer extension procedures that comprise steps of DNA meltingand of polymerase-mediated DNA synthesis, such as DNA sequencing and PCRprocedures, leading to high yields of dsDNA denaturation, namelyseparation of dsDNA into its two complementary strands at a lowertemperature, and high performance of DNA polymerases.

The concentration of the osmoprotectant to be used in a particularcycled primer extension reaction depends on the specific template DNA,the primer(s), the DNA polymerase and the reaction conditions employed.Low concentrations of THP(B) or proline, typically around 0.5-1.5M, arepreferred for lowering Tm of an average GC-content DNA, while higherconcentrations, typically 1-3M, are preferred for high GC-content DNA,so to further lower the Tm and hence the denaturing temperatureemployed. To avoid major dissociation of primer/template DNA, when highconcentrations of THP(B) (3-4M) and proline (4-5M) are used to lower DNATm to the range of 40-55° C. primers of at least 30 nucleotides areused. These modifications improve annealing and yield of the reaction.

The use of non-thermostable DNA polymerases such as T7 DNA polymerase orKlenow is of major importance in cases where accuracy of DNAamplification is crucial such as in detection of subtle changes in a DNAsequence and in processes of PCR typing and diagnosis of some geneticdiseases and cancer caused by minor mutations, due to their highfidelity in DNA replication and proofreading ability.

Performing primer extension reactions at reduced temperatures alsopermits the use of thermosensitive fluorescent and other labilecompounds for labeling newly synthesized DNA strands for use as probesin the detection of complementary target sequences of nucleotides bysensitive assays such as, chemiluminescence detection.

Reaction conditions used in PCR are variable depending on the nature ofthe template DNA and primers, and optimal pH and salt and magnesium ionsconcentrations are usually determined empirically for each particularreaction. A typical PCR procedure involves temperature cycling toprovide adequate conditions for accomplishing three steps in each PCRcycle: (i) DNA denaturation; (ii) primer annealing; and (iii) primerextension.

A standard denaturation incubation step (i) at 94-95° C. for 0.5-2 minis usually sufficient for separating DNA strands of an average GCcontent from the original and newly synthesized DNA. The primerannealing step (ii) is performed usually around 5° C. lower than themelting temperature of the primer-template DNA duplex. However, ifnon-specific PCR products are obtained in addition to the expectedproduct, the annealing temperature should be increased. The extension(step iii) of the annealed primer at its 3′ end to synthesize a new DNAstrand, complementary to the template strand, is usually carried out bythe thermostable enzyme Taq polymerase at 70-75° C., which is theoptimal temperature range for the enzyme activity (˜2-4 Kb/min.).

The complete denaturation of the DNA template, especially at the firstamplification cycles, is of most importance in PCR procedures, otherwiseits use as a template for the following reaction steps decreases andresults in poor yield of the PCR product. This is especially relevantwhen an amplified DNA duplex has a very high GC content, rendering itdifficult in strand separation, or when a target nucleotide sequence ispresent in a minute amount in the initial reaction mixture. Thus, PCRbuffers containing solutes leading to significant lower Tms of the DNAtemplates are most important in PCR procedures. The addition of proline,THP(B), THP(A) or mixtures thereof to PCR procedures is beneficial inthree levels: (i) increased yield of the amplified DNA products; (ii)increased sensitivity; and (iii) increased specificity of the reaction.The effect of proline and THP(B) in decreasing Tms of oligonucleotideprimers and of template DNAs, and the effect of proline, THP(B) andTHP(A) in stabilizing DNA polymerases, result in more efficient use ofthe template DNAs, primers and enzymes of the reaction, leading to highyield of PCR-amplified DNA product. Moreover, the increased sensitivityof PCR assays in the presence of the additive enables detection oftarget DNA sequences that are not detectable in its absence. This isespecially significant in cases where very rare or long target sequencesare to be amplified. In addition, the additives also improve the qualityof PCR amplification by reducing significantly or eliminatingnonspecific products. The improved accuracy of PCR in the presence ofproline, THP(B) and/or THP(A) enables performing PCR protocols withincreased number of cycles and longer cycle times, without impairing thequality of the reaction products.

In another embodiment, the invention provides a method for cycle DNAsequencing comprising contacting a template DNA with a primer homologousto a specific sequence on a target DNA in the presence of a DNApolymerase and an effective amount of proline, THP(B) and/or THP(A)under conditions allowing DNA sequencing.

A commonly used cycle DNA sequencing protocol known as Sanger or dideoxysequencing method, typically includes isolating double stranded templateDNA, separating it into its component single strands, adding asequencing primer homologous to a sequence of nucleotides on the targetDNA and performing a cycled primer extension of said primer on thetarget DNA. The cycled primer extension is performed in four paralleledreactions, each including a small amount of a dideoxynucleotidetriphosphate, either ddATP, ddCTP, ddGTP or ddTTP, along with a molarexcess of the four deoxynucleotide triphosphates (dNTPs) normallyrequired for DNA synthesis, i.e. dATP, dCTP, dGTP or dTTP. The growth ofthe extended DNA chain is stopped once a ddNTP molecule is incorporatedinto it, thus generating series of extension products of variouslengths. When these extension products of the four extension reactionsare separated side by side, for example on a polyacrylamide gel, apattern is obtained. By using a labeled primer or labeled ddNTP,typically radioactive or fluorescent, this pattern can be monitored, forexample, by autoradiography, fluorescence detectors etc, and the DNAsequence can be determined.

Cycle DNA sequencing also involves cycle primer extension, thus thesequencing outcome is influenced by similar criteria as mentioned abovefor PCR. The degree of template DNA and primer denaturation, as well asthe polymerase performance, are of crucial importance for thesensitivity and accuracy of a sequencing reaction. The exact reactionconditions for performing a cycle DNA sequencing method and theeffective concentrations of the added osmoprotectant vary depending onthe template DNA, primers, target DNA to be sequenced and the DNApolymerase used in a particular reaction.

Cycle sequencing performed in accordance with the invention in thepresence of proline, THP(B) and/or THP(A), is a beneficial and sensitivetool. The osmoprotectant additive permits obtaining a sequence of alonger stretch of nucleotides in a single reaction, as well as tosequence minute amounts of DNA present, for example, in limited samplesof blood or tissue used in forensic pathology and in evolutionarybiology. In addition, some GC-rich DNAs or other DNAs with complex orrigid secondary structure that are very difficult to sequence usingconventional reaction mixtures, can thus be successfully sequenced.Since in the presence of the additives the specificity of primerannealing is increased and non-specific extended products are mostlyeliminated, detection of rare mutations becomes feasible. This isespecially important in diagnosis of diseases characterized by a smallmutation in a gene nucleotide sequence or in identification of high CGGrepeats that are indicative of many human disorders, such asHuntington's disease (Han et al., 1994).

The kits for performing DNA amplification by PCR or for DNA cyclesequencing of the invention include, respectively, the reagentsnecessary for PCR or DNA sequencing (e.g. appropriate buffers, dNTPs,either Taq or a non-thermostable polymerase, etc.) and, in separatecontainers, THP(B) optionally with THP(A) or proline.

EXAMPLES

Materials and Methods

(i) Materials

For Examples 1-4, THP(A) and THP(B) were prepared according to Malin andLapidot (1996) and their water solutions were passed through a chelexcolumn to remove divalent cations before use. Betaine (Sigma) wasdissolved in water and passed through a chelex column before use. TaqDNA polymerase (recombinant) and Klenow fragment of DNA polymerase I (10units/μl) were purchased from MBI Fermentas, calf thymus DNA (used inthe DNA melting examples) and activated calf thymus DNA (used in thepolymerase activity assays), Micrococcus lysodeikticus DNA, Colstridiumperfringens DNA and poly(dA-dT) from Sigma. The oligonucleotides[d(ATGCAT)]₂ (SEQ ID NO: 1)and [d(GCTTAAGC)]₂ (SEQ ID NO: 2) and thefollowing 28-mer primers 1 and 2 were prepared by solid-phasephosphouramidate synthesis:

1. 5′>CGG GAT CCA TGG AAT ACG TAT ACG CTG C<3′(SEQ ID NO: 3)

2. 5′>CGG AAT TCT TAG CCG AAG AGT TCG CCG A<3′(SEQ ID NO: 4)

For Examples 5-9, L-proline 99+% and 99.5+% were purchased from Sigmaand from Fluka, respectively, and glycerol was from BDH. Activated calfthymus DNA and calf thymus DNA was from Sigma. Taq DNA polymerase(recombinant) and Klenow fragment of DNA polymerase I (10 units/μl) werepurchased from MBI Fermentas. Klentaql DNA polymerase from AB Peptidesand Pfu DNA polymerase (cloned) from Stratagene. Halobacteriummarismortui genomic DNA template was a generous gift of Dr. ShulamithWeinstein (Kimmelman Laboratory of Biocrystallization, WeizmannInstitute of Science).

Two pairs of primers were used in Examples 7-9: two 28-mer primers 3 and4 with 22 of complementary nucleotides each and with end restrictionsite: primer 3 containing BamHI restriction site and primer 4 containingEcoRI restriction site, and two 30-mer primers 5 and 6 with all 30complementary nucleotides:

3. 5′>CGG GAT CCA TGG AAT ACG TAT ACG CTG C<3′(SEQ ID NO: 3)

4. 5′>CGG AAT TCT TAG CCG AAG AGT TCG CCG A<3′(SEQ ID NO: 4)

5. 5′>ATG GAA TAC GTA TAC GCT GCA CTC ATC CTG<3′(SEQ ID NO: 5)

6. 5′>TTA GCC GAA GAG TTC GCC GAG GCC CTC ACC<3′(SEQ ID NO: 6)

All oligonucleotides and primers for Examples 1-9 were prepared by theChemical Service Unit of the Weizmann Institute of Science, Rehovot,Israel, and their solution concentrations were determined by UVabsorbance at 260 nm.

(ii) DNA Melting Experiments

DNA melting studies were conducted in a buffer (1 ml) containing 5.0 mMK₂HPO₄ and 0.1 mM Na₂EDTA at pH 7.5. The buffer and THP(B) or prolinesolutions were filtered through 0.22 μm Millipore membrane filter, priorto addition of the DNA, and then degassed with helium at roomtemperature. DNA samples were adjusted to O.D₃₆₀=0.2 and incubatedovernight at 37° C. before use, as previously described (Rees et al.,1993). DNAs in the above buffer with and without THP(B) or proline weremeasured in 1-cm path Teflon-stoppered quartz cell and incubated at theinitial assay temperature for 5 min. The increase in absorbance at 260nm was monitored in Hewlett Packard 9450A diode array spectrophotometerattached to a temperature programmer and controller. Both the sample andthe reference cells were heated together at a rate of 0.5° C./min andthe net absorbance was recorded after every 0.5° C. increase. The Tmswere determined graphically from the midpoints of the absorbance versustemperature profile.

(iii) NMR measurements of chemical shift changes

¹H NMR measurements were carried out on a Bruker AMX 400 NMR MHZspectrometer at 400.13 MHZ (equipped with an Aspect 300 control). Forthe ¹H NMR measurements, 1.0 mM DNA oligonucleotides were dissolved in0.5 ml phosphate buffer solution (pH 7.2) in D₂O (20 mM, for[d(ATGCAT)]₂ (SEQ ID NO:1) and 40 mM for [d(GCTTAAGC)]₂ (SEQ ID NO:2)containing 50 mM NaCl and 0.1 mM EDTA. The solutions were lyophilizedand then redissolved in 0.5 ml D₂O (99.96%), heated to 65° C. andgradually cooled to 5° C., and then degassed with argon at roomtemperature.

(iv) Klenow DNA polymerase activity assay

The assay was performed at 37° C. in 15 μl reaction mixture containing67 mM Tris-HCl (pH 7.4), 1.0 mM β-mercaptoethanol, 5.2 nM, [α-³²P]dATP,6.4 μM dATP and 320 μM of each dCTP, dGTP, dTTP, 0.6 mM activated calfthymus DNA, and 6.7 mM MgCl₂ for THP or 6.7, 10.0 and 15 mM MgCl₂ forproline, Klenow fragment (0.1 units) was added to the microtubes withreaction mixtures pre-heated at 37° C., and following 7.5 minutesincubation at 37° C. (a time-point within the region of linear kineticsdetermined in a separate experiment, not shown), the reaction microtubeswere placed on ice, and the reaction was stopped by addition of 12 μl of50 mM EDTA and then applied on strips of chromatographic paper (WhatmanNo. 3). Strips were washed three times by cold TCA 10%, dried and theradioactivity was counted.

(v) Determination of remaining activities of Tag polymerase afterincubation with THP(A) or THP(B) at elevated temperatures

Taq polymerase (0.5 units) was added to 50 μl buffer containing: 10 mMtris-HCl (pH 8.8 at 25° C.), 2.5 mg Halobacterium morismortui genomicDNA template, 2 μm of each of the dNTPs: dATP, dCTP, dGTP and dTTP, 0.12nM of each of the two 28-mer oligonucleotide primers 1 and 2 describedin section (i) above, 50 mM KCl, 0.08% Nonidet P40 and 1.0 mM MgCl₂.THP(B), THP(A) or glycerol were added from 3M stock solutions (pH 8.8 at25° C.). The reaction mixtures were overlaid with paraffin oil andincubated at 95° C. or 97° C. Aliquots (7.5 μl) were taken forpolymerase activity assay at different periods of time.

(iv) Determination of remaining activity of Klenow DNA polymerase afterincubation at 65° C. with proline

Klenow DNA polymerase (0.5 unit) was incubated at 65° C. in 50 μl buffercontaining: 67 mM tris-HCl (pH 7.4 at 25° C.), 2.5 ng Halobacteriummarismortui genomic DNA template, 4 μM of each of the dNTPs dATP, dCTP,dGTP and dTTP, 0.12 nM each of the two 28-mer oligonucleotide primers 3and 4 described in section (i) above, 6.7 mM MgCl₂ and either without orin the presence of 5.0M glycerol or proline. Tris-HCl buffer, templateDNA, dNTP, primers and MgCl₂ were added to PCR microtubes, evaporated todryness by speed-vacuum and respective volumes of water, proline (from a5.5M stock solution) or glycerol (from a 5.5M stock solution) wereadded. The microtubes were vortexed and Klenow enzyme was added to thesamples. Aliquots (5 μl) were taken for polymerase activity assay atdifferent periods of time as indicated in FIG. 11. The Klenow DNApolymerase activity assay was performed as described in section (iv)above at 6.7 mM concentration of MgCl₂. To each aliquot (5 μl) 20 μl ofstock solution containing all other components of the assay were added,making a total reaction volume of 25 μl and a 5-fold dilution of thealiquots. Thus, proline and glycerol concentration in the polymeraseassay were 1.0M, shown to be stimulative for Klenow polymerase activityin a separate experiment (data not shown).

(vii) Polymerase Chain Reaction (PCR) procedure with THP(B)

PCR was performed in 25 μl reaction mixture containing 3 ng temperatureDNA, 0.12 nM of each 28-mer oligonucleotide primer 1 and 2 described insection (i) above 0.5 units Taq DNA polymerase. 200 μM of each dNTP, inPCR buffer containing: 10 mM Tris-HCl (pH 8.8 at 25° C.), 50 mM KCl,0.08% Nonidet P40. MgCl₂ concentrations of 1.0 mM and 1.75 mM were usedin the absence and presence of THP(B), respectively, added from a 3Mstock solution (pH 8.8 at 25° C.). Reaction mixtures were overlaid withparaffin oil and preheated for 3 min at their respective denaturingtemperatures, except for mixtures of reactions performed at Td 95° C.,that were preheated for 3 min. at 94° C., and then subjected to 35thermal cycles as follows: (i) 30 sec incubation at 89-95° C., asindicated i each experiment (denaturation step); (ii) 90 sec. incubationat 55° C. (annealing step); and (iii) 60 sec incubation at 72° C.(primer extension).

(viii) PCR in the presence of proline, using Klenow DN4 polymerase.

PCR was performed in a 25 μl reaction mixture containing 100 ngHalobacterium marismortui genomic DNA template, 0.12 nM of each 30-meroligonucleotide primers 5 and 6 described in section (i) above 10 or 15units of Klenow DNA polymerase. 0.9 mM of each dNTP, in PCR buffercontaining 10 mM Tris-HCl (pH 7.4 at 25° C.) and 15 mM Mg(OAc)₂.Tris-HCl buffer, temperature DNA, dNTP, primers and Mg(OAc)₂ were addedto PCR microtubes, evaporated to dryness by speed-vacuum and dissolvedin 22 μl of a proline-glycerol solution (5.5M of L-proline in 12.5% w/vsolution of glycerol in water). Reaction mixtures were preheated for 3min at 75° C., and then subjected to 35 thermal cycles as follows: (i)20 sec incubation at 70° C. (denaturation step); (ii) 4 min incubationat 37° C. (primer annealing and primer extension steps). Klenow DNApolymerase (10 or 15 units) diluted up to 3 μl volume, containing 50%w/v glycerol, was added during the first primer annealing step at 37° C.

(ix) PCR in the presence of proline, using Tq DNA polymerase.

PCR was performed in 25 μl reaction mixture containing 3 ng ofHalobacterium marismortui genomic DNA temperature, 0.12 nM of each28-mer olignocleotide primers 3 and 4 described in section (i) above,0.5 units of Taq DNA polymerase, 200 μM of each dNTP, in PCR buffercontaining : 10 mM Tris-HCl (pH 8.8 at 25° C.), 50 mM KCl, 0.08% NonidetP40. MgCl2 concentrations of 1.0 mM and 1.8 mM were used in the absenceand in th presence of 1.0M L-proline, respectively. L-proline was addedfrom 5.5 M stock solution adjusted to pH 8.8 at 25° C. Reaction mixtureswere preheated for 3 min at their respective denaturation temperatures,except for reactions performed at Td 95° C., that were preheated for 3min at 94° C., and then subjected to 35 thermal cycles as follows: (I)30 sec incubation at 91-95° C., as indicated in each experiment(denaturation step); (ii) 90 sec incubation at 55° C. (primer annealingstep); and (iii) 60 sec incubation at 7° C. (primer extension).

(x) PCR in the presence of proline, using a mixture of Klentaql and Pfufor Vent) DNA polymerases.

PCR was performed in 25 μl reaction mixture containing 250 ng ofHalobacterium marismortui genomic DNA template, 0.12 nM of each 30-meroligonucleotide primers 5 and 6 described in section (i) above, 0.3 μlof Klentaql and Pfu (or Vent) enzymes mixture, prepared as described(Barnes, 1994), 200 μM of each dNTP, in PCR buffer containing : 10 mMTris-HCl (pH 8.3 at 25° C.) and 50 mM KCl Mg(OAc)₃ concentrations of 1.0mM and 14.5 mM were used in the absence and in the presence of 4.0ML-proline, respectively, L-proline was added from 5.5 M stock solutionadjusted to pH 8.3 at 25° C. Reaction mixtures were preheated for 1 minat their respective denaturation temperatures, except for reactionsperformed at Td 95° C., that were preheated for 1 min at 94° C., andthen subjected to 35 thermal cycles as follows: (I) 30 sec incubation at72-95° C., as indicated in each experiment (denaturation step); (ii) 90sec incubation at 37-55° C., as indicated in each experiment (primerannealing step); and (iii) 7 min incubation at 63-69° C., as indicatedin each experiment (primer extension).

Example 1 DNA melting in the presence of THP(B)

The effect o different concentrations of THP(B) on the melting profileof calf thymus DNA (42% GC) was studied. Melting experiments wereconducted as described in Materials and Methods, section (ii), in theabsence or presence of 0.8M, 2M, 3M and 4M THP(B).

As shown in FIG. 2, the addition of THP(B) significantly lowered the DNAmelting temperature and sharpened its transition profile. The DNAmelting temperature in aqueous solution, 62° C., was lowered to 41° C.in the presence of 3 or 4M THP(B).

The effect of THP(B) on DNA melting temperatures was examined on otherDNAs with different base compositions, such as Micrococcus lysodeikticusand Clostridium perfringens DNAs (72% and 26% GC, respectively) and onthe synthetic poly(dA-dT). As shown in FIG. 3, the melting temperatures(Tm) of the different DNAs decreased with the increase of THP(B)concentration in the incubation mixture. This effect is more pronouncedfor GC-rich DNAs. While the oligonucleotide poly(dA-dT) did not exhibitany change in the melting temperature in the presence of 1-4M (THP(B),3-4M THP(B) eliminated the base-pair composition dependence of DNAthermal melting. As shown in FIG. 3, in the presence of 4M THP(B), allDNAs with a wide range of GC content melt in a very narrow temperaturerange (40-43° C.), while in the absence of THP(B), the meltingtemperatures ranged from 39 to 75° C. This isostabilization effect byTHP(B) may be explained as a result of greater destabilization ofGC-rich than AT-rich DNAs.

Example 2 Short oligonucleotides melting in the presence of THP(B)

The thermal transitions of the short oligonucleotides [d(ATGCAT)]₂ (SEQID NO:1) (FIG. 4A) and [d(GCTTAAGC)]₂ (SEQ ID NO:2) (FIG. 4B) werestudied in the absence (open squares) and presence of 0.5M (opencircles) and 1.0M (filled squares) THP(B). NMR chemical shift changes ofthe C4H5 proton of [d(ATGCAT)]₂ (SEQ ID NO:1) and of the G1H8 proton of[d(GCTTAAGC)], (SEQ ID NO:2) were measured as a function of increasingtemperatures as described in Materials and Methods section (iii). Theresults of these experiments are depicted in FIGS. 4A-B and summarizedin Table 1.

TABLE 1 Tm° C. DNA-THP (B) DNA-betaine Oligonucleotide DNA 0.5 M 1.0 M1.0 M [d(ATGCAT)]₂ (SEQ ID NO:1) 31.5 29.5 28.0 29.2 [d(GCTTAAGC)]₂ (SEQID 48.0 45.0 42.0 — NO:2)

As shown in Table 1, the melting temperatures of [d(ATGCAT)]₂ (SEQ IDNO:1) and of [d(GCTTAAGC)]₂ (SEQ ID NO:2) decreased by 2° C. and by 3°C., respectively, in the presence of 0.5M THP(B), and by 3.5° C. and 6°C., respectively, in the presence of 1.0M THP(B). Data were compared tothe melting temperature of [d(GCTTAAGC)]₂ (SEQ ID NO:2) in the presenceof betaine. The decrease in Tm by betaine was only −2° C. at 1.0Mconcentration, about two-fold higher concentration of betaine is neededfor exerting the same Tm decline caused by THP(B).

Example 3 THP(B) and THP(A) effects on Taq DNA polymerase stability atelevated temperatures

The effect of THP(B) and THP(A) on the remaining activity of Taq DNApolymerase incubated at elevated temperatures for different periods oftime were studied.

After 90 min incubation at 95° C., Taq DNA polymerase was only 30%active. The enzyme was remarkably stabilized upon addition of eitherTHP(B) or THP(A). After incubation at 95° C. in the presence of 0.5MTHP(B) or 0.5M THP(A), the half life of Taq polymerase was 70 min and60-90 min, respectively, in comparison to the half life of 30-40 minobserved in the absence of additive (not shown). Comparable protectiveeffects were obtained when Taq DNA polymerase was incubated at 95° C. inthe presence of a combination of THP(A) and THP(B) (results not shown).Thus, THP(B) and/or THP(A) present in the reaction mixture enabledoubling PCR cycles without increased loss of enzyme activity.

in FIG. 5 are shown results of similar experiments measuring the thermalinactivation of Taq polymerase at 97° C. in the absence (filled circles)or presence of 1M THP(B) (filled squares), 1M THP(A) (open squares) incomparison to 1M glycerol (open diamonds). The thermal inactivation ofthe enzyme at the elevated temperature 97° C. was, as expected, morerapid than the inactivation at 95° C., almost a complete loss (>95%) ofenzyme activity was observed following 60 min incubation at 97° C., withno additives. However, also the protective effects by THP(B) and THP(A)were more dramatic: the remaining Taq polymerase activities, following30 min incubation at 97° C. were 40% and 50% in the presence of 1MTHP(B) or THP(A), respectively, in comparison to 10% remaining activityin the absence of additives. As a result of 60 min incubation at 97° C.,the remaining Taq polymerase activity in the absence of additive was 5%,whereas in the presence of 1M THP(B) or THP(A) the remaining activitieswere 20% and 45%, respectively. The results shown in FIG. 5 indicatethat THP(A) is more effective than THP(B) or glycerol in stabilizing TaqDNA polymerase.

Example 4 PCR in the presence of THP(B)

The combined effect of THP(B) on DNA melting temperatures and on Taq DNApolymerase activity and stability at elevated temperatures was followedunder PCR conditions.

PCR reaction was performed by Taq DNA polymerase as described inMaterials and Methods, section (viii), using as a temperature wholegenomic DNA of Halobacterium marismortui (66.5% GC) and the 28-merprimers 1 and 2 described in section (i).

In FIG. 6 are depicted the amplified DNA sequences produced by PCRperformed at 95° C. and 92° C., in the absence and presence of 0.5MTHP(B), respectively, showing that yield and specificity of the DNAamplification was improved by the presence of THP(B). At 92° C.amplified sequences were produced only in the presence of THP(B), butnot in its absence.

The effect of 1.0M THP(B) in the PCR buffer mixture in presented inFIGS. 7A-C. A “control” assay was performed at Td 95° C. in the absenceand presence of 1.0M THP(B) with two concentrations of Taq DNApolymerase, 0.5 and 0.75 units in 25 μl PCR reaction mixture. Thepresence of 1.0M THP(B) improved PCR specific amplification at Td 95° C.(FIG. 7A). However, the most significant results were obtained whendenaturation temperatures of the DNA were reduced from 95° C. to 90° C.(FIG. 7B) in the presence of 1.0M THP(B) (either with 0.5 or 0.75 unitsof Taq DNA polymerase in 25 μl reaction mixture). Under theseconditions, specific amplified sequence was generated only in thepresence of THP(B), while no trace of amplified DNA could be detected inthe absence of this additive.

When Td was further lowered to 89° C. in the presence of 0.5 units TaqDNA polymerase in 25 μl reaction mixture, amplified DNA sequence wasmarkedly lower, even in the presence of 1.0M THP(B) but no trace ofamplified DNA was detected in the absence of THP(B) (FIG. 7C).

Example 5 DNA melting in the presence of proline.

The effect of different concentrations of proline on the melting profileof calf thymus DNA (42% GC) was studied. Melting experiments wereconducted as described in Materials and Methods, section (ii), in theabsence or presence of 2M, 3.5M 5M and 6.2M proline.

As shown in FIG. 8A, the addition of proline significantly lowered theDNA melting temperature and sharpened its transition profile. The DNAmelting temperature in aqueous solution, 62° C., was lowered in 27° C.in the presence of 6.2M proline.

The effect of proline on DNA melting temperatures was examined ondifferent DNAs with different base compositions, such as Micrococcuslysodeikticus and Clostridium perfringens DNAs (72% and 26% GC,respectively), calf thymus DNA (42% GC) and on the syntheticpoly(dA-dT). As shown in FIG. 8B, the melting temperatures (Tm) of thedifferent DNAs decreased in the presence of 6.2M proline concentrationin the incubation mixture. The range of melting DNA with GC content of72% is about 5° C. higher than that of GC content of 42% and 26%, whilepoly(dA-dT) melts about 15° C. lower.

The effect of increasing concentration of proline as depicted in FIG. 9Aon the four DNAs reveals that the effect was pronounced for GC-richDNAs. While the oligonucleotide poly(dA-dT) did not exhibit any changein the melting temperature in the presence of 1-5M proline, a smalleffect occurred in the range of 6-6.2M proline. Proline at 6.2M almosteliminated the base-pair composition dependence of DNA thermal melting.As shown in FIG. 9A, in the presence of 6.2M proline all DNAs with awide range of GC content melt in a very narrow temperature range (25-32°C.), while in the absence of proline the melting temperatures rangedfrom 38 to 78° C.

FIG. 9B depicts changes in dTm/dGC as a function of prolineconcentration. A linear correlation is presented for prolineconcentration of up to 5M.

Example 6 Klenow polymerase activity in the presence of proline.

To study the effect of 5.0M L-proline on the Klenow DNA polymeraseactivity, experiments were conducted as described in Materials andMethods, section (iv), in the presence of different concentrations ofMgCl₂: 6.7 mM, 10.0 mM and 15.0 mM MgCl₂.

As shown in FIG. 10, L-proline only slightly decreased Klenow DNApolymerase activity. The activity of the enzyme remained high enough,particularly at 10.0 (hatched bar, middle) and 15.0 mM MgCl₂ (hatchedbar, right).

Example 7 The effect of 5.0M proline on the stability of Klenow DNApolymerase at 65° C.

The remaining activity of Klenow DNA polymerase incubated at 65° C. inthe presence of 5.0M proline, 5.0M glycerol or without any additives,was measured as described in Materials and Methods, section (vi). Asshown in FIG. 11, Klenow DNA polymerase at 65° C. has a half-life ofless than one minute with no additives (filled circles), 3 minutes inthe presence of 5.0M glycerol (filled triangles) and 21 minutes in thepresence of 5.0M proline (open diamonds).

Example 8 PCR in the presence of proline, using Klenow DNA polymerase.

The combined effects of proline in Klenow DNA polymerase stability atelevated temperatures and on DNA denaturation temperature step,permitted a successful design of cycled PCR conditions for this enzyme.PCR was performed by Klenow DNA polymerase as described in Materials andMethods, section (viii).

PCR amplification of a 349 b.p. fragment (66.5% GC) of Halobacteriummarismortui genomic DNA (from position 2546 to 2843) was performed in a25 μl reaction mixture containing 100 ng of the DNA template, 0.12 nM ofeach 30-mer oligonucleotide primers 5 and 6 described in section (i)above, 0.9 mM of each dNTP, 10 mM Tris-HCl (pH 7.4 at 25° C.) and 15 mMof magnesium acetate. Tris-HCl buffer, template DNA, dNTP, primers andmagnesium acetate were added to PCR microtubes from stock solutions,evaporated to dryness by speed-vacuum and dissolved in 22 μl of aproline-glycerol solution, containing 5.5M of L-proline in a 12.5% v/wsolution of glycerol in water. Klenow polymerase (10 units/μl, storagebuffer contains 50% w/v glycerol) and, in order to keep constantglycerol concentration in the PCR mixtures, aliquots of glycerolsolution in water (50% w/v glycerol) were added during the first primerannealing step.

As shown in FIG. 12, in lanes 1, and 2, 1.0 μl of Klenow polymerase (10units) and 2.0 μl of the glycerol solution were added, and in lanes 3,and 4, 1.5 μl of Klenow polymerase (15 units) and 1.5 μl of the glycerolwere added. The final concentration of L-proline in all PCR mixtures was4.85M and of glycerol was 17% w/v. All PCR reactions were run on a MJResearch PTC-100 machine equipped with a normal block (ramping rate is1° C. per section). Reaction mixtures were preheated for 3 min at 75°C., and then subjected to 35 thermal cycles as follows: a) 20 secincubation at 70° C.; b) 4 min incubation at 37° C. Reaction productswere run on a 2% agarose gel and strained by ethidium bromide.

The results shown in FIG. 12 reveal that proline concentration i therange of 3-5.5M is sufficient to confer stability to Klenow DNApolymerase and to conduct a successful PCR protocol.

Example 9 PCR in the presence of proline, using Taq DNA polymerase.

FIG. 13 shows PCR, using Taq DNA polymerase, performed in the absenceand in the presence of 1.0M proline, as described in Materials andMethods, section (ix). Addition of 1.0M proline to the reaction mixturedid not impair PCR performance at denaturation temperature 95° C. andenabled successful PCR at decreased denaturation temperature, namely 91°C.

Example 10 PCR in the presence of proline, using mixture of Klentaql andPfu DNA polymerase.

PCR was performed in the presence of 4.0M proline, using a mixture ofKlentaql and Pfu DNA polymerases, as described in Materials and Methods,section (x). Reaction mixtures were preheated for 1 min at theirrespective denaturation temperatures (77° C. and 75° C.), and thensubjected to 35 thermal cycles as follows: (i) 30 sec incubation at 77°C. or 75° C. (denaturation step), (ii) 90 sec incubation at 44° C.(primer annealing step); and (iii) 7 min incubation at 65° C. (primerextension).

As shown in FIG. 14, there is a clear correlation between theconcentration of proline in the mixture and the minimal denaturationtemperature. Thus, true for above mentioned conditions, 4.0Mconcentration of proline was enough for successful PCR at the 77° C.denaturation temperature, but not at 75° C.

References

1. Barnes, W. M. (1994) PCR amplification of up to 35-kb DNA with highfidelity and high yield from λ bacteriophage temperatures, Proc. Natl.Acad. Sci. 91, 2216-2220.

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6 1 12 DNA Artificial Sequence Synthetic 1 atgcatatgc at 12 2 16 DNAArtificial Sequence synthetic 2 gcttaagcgc ttaagc 16 3 28 DNA ArtificialSequence synthetic 3 cgggatccat ggaatacgta tacgctgc 28 4 28 DNAArtificial Sequence synthetic 4 cggaattctt agccgaagag ttcgccga 28 5 30DNA Artificial Sequence synthetic 5 atggaatacg tatacgctgc actcatcctg 306 30 DNA Artificial Sequence synthetic 6 ttagccgaag agttcgccgaggccctcagg 30

What is claimed is:
 1. A method for performing a cycled primer extensionreaction comprising the steps of: (i) contacting a template DNAcomprising a target sequence of nucleotides with at least one primeroligonucleotide complementary to a nucleotide sequence at the 3′-end ofsaid target sequence, under conditions allowing annealing of said priorto its complementary nucleotide sequence on said target sequence, in thepresence of an osmoprotectant selected from2-methyl-4-carboxy-3,4,5,6-tetrahydropyrimidine and mixtures of THP(B)and proline, to lower the melting temperature of said template DNAand/or of said primer; and (ii) carrying out a polymerase-mediatedextension of said primer on said target sequence of nucleotides in thepresence of an osmoprotectant selected from THP(B),2-methyl-4-carboxy-5-hydroxy-3,4,5,6-tetrahydropyrimidine and mixturesof THP(B), THP(A), and/or proline to stabilize said polymerase; thusobtaining a high yield specific extension of the primer on said targetsequence of nucleotides of the template DNA.
 2. The method according toclaim 1 wherein steps (i) and (ii) are repeated a plurality of times,and each step (i) is preceded by DNA thermal denaturation at atemperature suitable for separating both said template DNA into itsstrands and the polymerase-extended primer of step (ii) from itscomplementary target sequence of nucleotides, said temperature being atemperatures in which the polymerase used in step (ii) is stable.
 3. Themethod according to claim 1 for determining a nucleotide sequence of atarget DNA, wherein in step (i) the target sequence of the template DNAis a sequence of nucleotides to be sequenced, and thepolymerase-mediated extension of the primer in step (ii) is carried outin the presence of dATP, dCTP, dGTP and dTTP, and in the presence of aminute amount of either ddATP, ddCTP, ddGTP or ddTTP, prior to thedetermination of the nucleotide sequence of the target DNA.
 4. Themethod according to claim 3 wherein dGTP is replaced by 7-deaza-dGTP. 5.A method for determining a nucleotide sequence of a target DNAcomprising: (i) heating a template DNA comprising a target sequence ofnucleotides to be sequenced at a temperature suitable for separatingsaid template DNA into its strands in the presence of an osmoprotectantselected from 2-methyl-4-carboxyl-3,4,5,6-tetrahydropyrimidine andmixtures of THP(B) and proline; (II) contacting said denatured templateDNA of step (i) with a primer oligonucleotide complementary to anucleotide sequence at the 3′-end of said target sequence of nucleotidesunder conditions allowing annealing of said primer to its complementarynucleotide sequence on the target sequence, in the presence of anosmoprotectant selected from2-methyl-4-carboxy-3,4,5,6,-tetrahydropyrimidine and mixtures of THP(B)and proline; (III) carrying out a polymerase-mediated extension of saidprimer of step (ii) in the presence of dATP, dCTP, dGTP and dTTP, of aminute amount of either ddATP, ddCTP, ddGTP or ddTTP and of anosmoprotectant selected from THP(B),2-methyl-4-carboxy-5-hydroxy-3,4,5,6-tetrahydropyrimidine and mixturesof THP(B), THP(A) and/or proline; (iv) repeating steps (i)-(iii) aplurality of times; and (v) determining the nucleotide sequence of thetarget DNA.
 6. The method according to claim 5 wherein dGTP is replacedby 7-deaza-dGTP.
 7. The method according to claim 1 for amplifying atarget sequence of nucleotides by polymerase chain reaction (PCR),wherein in step (i) the target sequence of the template DNA is asequence of nucleotides to be amplified and the template DNA iscontacted with two oligonucleotide primers complementary to thenucleotide sequences at the 3′-ends of said target sequence ofnucleotides and its opposite strand; in step (ii) a polymerase-mediatedextension of the annealed primers of step (i) is carried out; steps(i)-(ii) are repeated a plurality of times, the last step being step(ii); thus generating multiple copies of the target sequence ofnucleotides.
 8. A method for amplifying a target sequence of nucleotidesby polymerase chain reaction (PCR) comprising the steps of: (i) heatinga template DNA comprising a target sequence of nucleotides to beamplified at a temperature suitable for separating said template DNAinto its strands in the presence of an osmoprotectant selected from2-methyl-4- carboxy-3,4,5,6-tetra-hydropyrimidine and mixtures of THP(B)and proline; (ii) contacting the template DNA of step (i) with twooligonucleotide primers complementary to nucleotide sequences at the3′-ends of said target sequence of nucleotides and its opposite strand,in the presence of an osmoprotectant selected from2-methyl-4-carboxy-3,4,5,6-tetrahydropyrimidine and mixtures of THP(B)and proline, under conditions allowing annealing of said oligonucleotideprimers to their complementary sequences on said target sequence ofnucleotides and its opposite strand; (iii) carrying out apolymerase-mediated extension of the annealed primers of step (ii) inthe presence of an osmoprotectant selected from THP(B),2-methyl-4-carboxy-5-hydroxy-3,4,5,6-tetrahydropyrimidine and mixturesof THP(B), THP(A) and/or proline; and (iv) repeating steps (i)-(iii) aplurality of times, the last step being step (iii), thus generatingmultiple copies of the target sequence of nucleotides.
 9. The methodaccording to claim 1 wherein said template DNA is a GC-rich DNA.
 10. Themethod according to claim 1 wherein the polymerase in step (ii) or (iii)is a thermostable DNA polymerase.
 11. The method according to claim 8wherein said thermostable DNA polymerase is Taq polymerase, Klentaql DNApolymerase or Pfu polymerase.
 12. The method according to claim 1wherein the polymerase in step (ii) or (iii) is a non-thermostable DNApolymerase.
 13. The method according to claim 12 wherein saidnon-thermostable DNA polymerase is selected from T7 DNA polymerase, T4DNA polymerase, Klenow fragment of DNA polymerase I, reversetranscriptases, Bca DNA polymerase, Bst DNA polymerase and mutants ofthese polymerases.
 14. A method for lowering the melting temperature ofdouble-stranded DNA (dsDNA) comprising adding to the incubation mixtureof said dsDNA an amount of2-methyl-4-carboxy-3,4,5,6-tetrahydropyrimidine, effective for loweringthe melting temperature of double-stranded DNA.
 15. A method forincreasing stability of a DNA polymerase of elevated temperaturescomprising adding to the incubation mixture of said polymerase an amountof an osmoprotectant selected from2-methyl-4-carboxy-3,4,5,6-tetrahydropyrimidine,2-methyl-4-carboxy-5-hydroxy-3,4,5,6-tetrahydropyrimine (THP(A)), andmixtures of THP(B), THP(A) and/or proline, effective for increasing thethermal stability of DNA polymerases of elevated temperatures.
 16. A kitfor determining a nucleotide sequence of a target DNA according to themethod of claim 3 comprising in separate containers: (a) the reagentsnecessary for DNA sequencing, and (b)2-methyl-4-carboxy-3,4,5,6-tetrahydropyrimidine, abbreviated as THP(B),or a mixture of THP(B) and2-methyl-4-carboxy-5-hydroxy-3,4,5,6-tetrahydropyrimine, abbreviated asTHP(A).
 17. A kit for amplifying a target sequence of nucleotidesaccording to the method of claim 7 comprising in separate containers:(a) reagents necessary for a polymerase chain reaction, and (b)2-methyl-4-carboxy-3,4,5,6-tetrahydropyrimidine, abbreviated as THP(B),or a mixture of THP(B) and2-methyl-4-carboxy-5-hydroxy-3,4,5,6-tetrahydropyrimine, abbreviated asTHP(A).